This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-247729, filed Aug. 17, 2000, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to high estrogen-sensitive medaka fish, and more specifically, to transgenic medaka fish having a medaka-derived estrogen receptor gene introduced.
The transgenic medaka fish of the present invention can be used to detect an estrogen-like endocrine disrupting chemicals, and also used as an experimental animal for elucidating development mechanism of thrombosis and as bioassay system for developing a therapeutic agent of thrombosis.
2. Description of the Related Art
Recently, the effects of chemical substances present in the environment upon endocrine system of an organism have been intensively studied, and have attracted a high interest increasingly. Since many chemical substances having endocrine disrupting activity upon an organism exhibit estrogen-like action, most of studies are directed to estrogen which is a female hormone.
Pollution of rivers with such estrogen-like chemical substances has constituted a social problem on a global scale. To detect the estrogen-like chemical substances from a river in the present invention, we tried the use of medaka fish which has excellent features as an experimental animal. However, the medaka fish cannot be used as an aquatic animal for testing environmental water, since it is not sensitive even to an extremely small amount of estrogen. This problem was found for the first time by the present inventor in the course of making the present invention.
The present invention was made to overcome the aforementioned problem. An object of the present invention is to provide transgenic medaka fish having sensitivity to a very small amount of estrogen. Another object of the present invention is to provide a method of producing medaka fish having one or more thrombi by using the transgenic medaka fish, and to provide the medaka fish having one or more thrombi produced by the method. A further object of the present invention is to provide a method of testing an estrogen-like acting substance by using the transgenic medaka fish.
To attain the aforementioned objects, the present inventor have succeeded in preparing transgenic medaka fish having a medaka-derived estrogen receptor gene introduced, namely, high estrogen-sensitive medaka fish.
The present invention was made based upon such achievement.
To be more specific, the present invention was achieved by the means described below.
(1) A polynucleotide having a nucleotide sequence represented by Sequence ID No: 1.
(2) A polynucleotide comprising the nucleotide sequence from 211 to 1935 position represented by Sequence ID No: 1.
(3) A protein having an amino acid sequence encoded by the polynucleotide described in (2).
(4) A recombinant vector containing the polynucleotide described in (1) or (2).
(5) Transgenic medaka fish into which the polynucleotide described in (1) or (2) is introduced.
(6) A method of producing medaka fish having one or more thrombi, comprising the step of raising the transgenic medaka fish described in (5) in the presence of estrogen.
(7) Medaka fish having one or more thrombi, which is obtained by raising the transgenic medaka fish described in (5) in the presence of estrogen.
(8) A method of testing an estrogen-like action in test water, comprising the steps of:
raising the transgenic medaka fish described in (5) in the test water; and
observing whether or not one or more thrombi are formed in the medaka fish after the raising step.
(9) The method described in (8), wherein the test water is water taken from environment.
(10) The method described in (8), wherein the test water is water having a test substance added.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
Now, the present invention will be explained in detail.
In the present invention, the medaka fish to be used in cloning an estrogen receptor gene and the medaka fish to be used in introducing the cloned gene are not particularly limited, as long as they belong to a species Oryzias latipes. The medaka fish actually used in the present invention were obtained from the BioScience Center, Nagoya University. The obtained medaka fish were grown while feeding Tetramin (Tetra) in an amount of about 1-10 mg/day.
It should be noted that fertilized eggs of the medaka fish cannot be deposited, because a technology for resuming the development of a freeze-stored egg, as the need arises, has not yet been established. Therefore, the medaka fish used in the present invention are now raised under control of the present inventor with responsibility. As described in the above, the medaka fish to be used in the present invention are not limited to those raised by the present inventor, and any medaka fish may be used in the present invention.
{Cloning of Medaka-Derived Estrogen Receptor Gene}
The medaka-derived estrogen receptor gene of the present invention is cloned from liver cDNA library of adult medaka fish. More specifically, the medaka-derived estrogen receptor cDNA is cloned by preparing a probe based on a nucleotide sequence of a human-derived estrogen receptor gene and screening the above cDNA library by use of the probe.
The nucleotide sequence of the cloned estrogen receptor cDNA is determined, and the amino acid sequence predicted from the nucleotide sequence is determined. The nucleotide sequence of the medaka-derived estrogen receptor cDNA and the amino acid sequence are shown by Sequence ID No: 1 and Sequence ID No: 2, respectively in the Sequence Listing.
In the present invention, the nucleotide sequence for expressing medaka estrogen receptor may have arbitrary length, as long as it comprises at least a coding region (i.e., the nucleotide sequence from 211 to 1935 position represented by Sequence ID No: 1). Furthermore, in the present invention, the amino acid sequence of the medaka estrogen receptor may also have deletion, addition and/or substitution of one or several amino acids in the amino acid sequence represented by Sequence ID No: 2, as long as it has the same function as the protein consisting of the amino acid sequence represented by Sequence ID No: 2.
{Preparation of Recombinant Vector}
The medaka-derived estrogen receptor cDNA cloned by the aforementioned method is introduced into a vector. The introduction of the cDNA into a vector can be performed in accordance with a known genetic engineering process. In this manner, it is possible to prepare a recombinant vector into which the medaka-derived estrogen receptor gene is inserted.
The vector to be used in the present invention is not particularly limited, as long as it can express the protein encoded by a foreign gene inserted therein. In the present invention, it is preferable to use a plasmid having a promoter sequence and a poly (A) signal sequence, as a vector. For example, as described later in examples, a new plasmid vector is constructed by purifying each DNA fragment from another plasmid containing a DNA fragment of a medaka actin promoter and from another plasmid containing a DNA fragment of a SV40 poly (A) signal, and the resultant plasmid vector may be used.
{Preparation of Transgenic Medaka Fish}
The recombinant vector prepared as mentioned above is transferred into a nucleus of a medaka fertilized egg, thereby preparing transgenic medaka fish capable of expressing the estrogen receptor gene in an excessive amount. As the medaka fertilized egg to be transformed in the present invention, an embryo at a single-cell stage or a two-cell stage within one hour after fertilization is preferable. The recombinant vector can be transferred into the fertilized egg by means of a known microinjection procedure.
The fertilized eggs which have been subjected to the gene transfer operation are preferably hatched in a medaka physiological saline solution (7.5 g/L NaCl, 0.2 g/L KCL, 0.2 g/L CaCl2, 0.02 g/L NaHCO3) at 25-28xc2x0 C. The hatched medaka fish are raised for about 4 months until they become adult fish.
From the adult fish, the fish actually having the gene introduced are screened by the following method. A DNA is first extracted from the adult fish. Then, from the extracted DNA, the estrogen receptor gene is amplified by a PCR, and the amplified DNA fragment is subjected to electrophoresis. The estrogen receptor gene inherently present in the chromosome of wild medaka fish has intron, whereas the estrogen receptor gene introduced into the medaka fish herein is a cDNA having no intron. Therefore, two types of estrogen receptor genes can be distinguished by electrophoresis on the basis of the difference of the size of DNA bands. By such procedure, it is possible to screen the medaka fish having the gene successfully introduced. However, it is not clear whether or not the estrogen receptor gene is introduced into the chromosome of a germ cell (sperm or egg) of the medaka fish screened in this step.
Subsequently, the medaka fish having the estrogen receptor gene introduced into the chromosome of a germ cell are screened by the following method. The medaka fish which has been confirmed to have the gene introduced by the aforementioned method is crossed with wild medaka fish, thereby obtaining offspring. If the medaka fish inheriting the estrogen receptor gene introduced is identified among the offspring, its parent turns out to be desired medaka fish having the estrogen receptor gene introduced in the chromosome of a germ cell. Whether or not the medaka offspring inherits the introduced gene can be identified, in the same way, by electrophoresis on the basis of the difference of the size of DNA bands between the introduced gene and the inherent gene.
The screened medaka fish having the estrogen receptor gene introduced in the chromosome can inherit the gene from generation to generation. Such medaka fish corresponds to a desired transgenic medaka fish of the present invention.
The expression of the estrogen receptor gene in the transgenic medaka fish of the present invention was checked by RT-PCR, and it was confirmed that the estrogen receptor gene was actually expressed. On the other hand, the expression of the estrogen receptor gene was rarely detected in wild medaka fish. As described above, the transgenic medaka fish of the present invention has a large number of estrogen receptors by the expression of the introduced gene. For this reason, the transgenic medaka fish of the present invention shows sensitivity even to a very small amount of estrogen.
{Preparation of Medaka Fish Having One or More Thrombi}
In the present invention, it was found that the medaka fish having one or more thrombi can be prepared, even if the transgenic medaka fish of the present invention is raised in the presence of estrogen whose concentration is lower than that capable of inducing the formation of a thrombus in wild medaka fish.
As the transgenic medaka fish to be used for the formation of a thrombus, it is preferable to employ an embryo within 12-24 hours after fertilization, because the formation of a thrombus in the embryo can be easily observed under a dissecting microscope. To form a thrombus in the transgenic medaka fish of the present invention, it is sufficient to contain estrogen in the concentration of 10-20 ng/L, preferably 100-200 ng/L, in the environment for raising medaka fish. It is noted that wild medaka fish require estrogen in the concentration of 4 mg/L or more in order to form a thrombus. As the term of raising the medaka fish in the presence of estrogen, a period of three to four days is necessary for the formation of a thrombus. The growth conditions may be the same as those generally employed for raising medaka fish.
It is possible to confirm the formation of a thrombus under a dissecting microscope.
The medaka fish having one or more thrombi prepared in this manner is useful for studying therapy of thrombosis.
{Method of Testing an Estrogen-Like Acting Substance}
The presence or absence of the estrogen-like acting substance can be detected by growing the transgenic medaka fish of the present invention in test water to be checked for the estrogen-like action and then observing whether or not a thrombus is formed in the medaka fish.
As the transgenic medaka fish to be used in order to detect an estrogen-like acting substance, an embryo within 12-24 hours after fertilization is preferable because it is easily observed under a dissecting microscope.
The xe2x80x9ctest waterxe2x80x9d used herein may be either water collected from the environment (river etc.), which may perhaps contain an estrogen-like acting substance, or water added a chemical substance, which may perhaps act like estrogen, as a test substance. When the former water taken from the environment is used, it is possible to detect the presence or absence of the estrogen-like acting substance in the environmental water. When the latter water added a test substance is used, the estrogen-like action of the test substance can be checked.
When the test substance is added to the test water, it is preferable to set a concentration thereof at 10 ng/L to 1 mg/L. As the period for raising the medaka fish in the test water, 3-6 days are appropriate. The growth conditions may be the same as those usually raising medaka fish.
It is possible to observe the formation of a thrombus under a dissecting microscope.
In fact, an estrogen-like acting chemical substance is known to have an endocrine disrupting action even in an extremely small amount. Therefore, the transgenic high estrogen-sensitive medaka fish of the present invention is very useful in both the aforementioned tests of the environmental water and the test substance.